WO2014114702A1 - Microscope optique et procédé de microscopie - Google Patents

Microscope optique et procédé de microscopie Download PDF

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Publication number
WO2014114702A1
WO2014114702A1 PCT/EP2014/051301 EP2014051301W WO2014114702A1 WO 2014114702 A1 WO2014114702 A1 WO 2014114702A1 EP 2014051301 W EP2014051301 W EP 2014051301W WO 2014114702 A1 WO2014114702 A1 WO 2014114702A1
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WO
WIPO (PCT)
Prior art keywords
light
sample
optical
illumination
detector
Prior art date
Application number
PCT/EP2014/051301
Other languages
German (de)
English (en)
Inventor
Wolfgang Bathe
Ralf Netz
Original Assignee
Carl Zeiss Microscopy Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Carl Zeiss Microscopy Gmbh filed Critical Carl Zeiss Microscopy Gmbh
Priority to EP14701716.4A priority Critical patent/EP2948810B1/fr
Priority to CN201480005722.4A priority patent/CN104956249B/zh
Priority to US14/762,930 priority patent/US9671600B2/en
Priority to JP2015554140A priority patent/JP6286449B2/ja
Publication of WO2014114702A1 publication Critical patent/WO2014114702A1/fr
Priority to US15/442,937 priority patent/US9989746B2/en

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Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0072—Optical details of the image generation details concerning resolution or correction, including general design of CSOM objectives
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0036—Scanning details, e.g. scanning stages
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0036—Scanning details, e.g. scanning stages
    • G02B21/0044—Scanning details, e.g. scanning stages moving apertures, e.g. Nipkow disks, rotating lens arrays
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/02—Objectives
    • G02B21/025—Objectives with variable magnification
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/58—Optics for apodization or superresolution; Optical synthetic aperture systems
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/008—Details of detection or image processing, including general computer control

Definitions

  • the present invention relates to a light microscope according to the preamble of claim 1.
  • the invention is directed to a microscopy method according to the preamble of claim 13.
  • a generic light microscope has a sample plane in which a sample to be examined can be positioned, a light source for emitting illumination light, optical imaging means for directing the illumination light into the sample plane, and a detector device for detecting sample light coming from the sample adjacent detector elements have a distance to one another which is smaller than an Airy disk, which generates a point in the sample plane on the detector device.
  • Electronic means can determine an image of the sample based on the detected sample light.
  • a generic microscopy method for examining a sample which is positioned in a sample plane of a light microscope
  • illumination light is directed into the sample plane, that the illumination light is moved over the sample plane as illumination scanning movement and that the sample light coming from the sample is detected by a detector device which has a plurality of detector elements, wherein adjacent detector elements have a distance to one another which is smaller than an Airy disk, which generates a point in the sample plane on the detector device.
  • Electronic means can thereby determine an image of the sample based on the detected sample light.
  • detector elements are used in the generic light microscope and the microscopy method, which are smaller than an Airy disk, which generates a point in the sample plane on the detector device.
  • the Airy is defined by the first zeros of the rotationally symmetric light distribution of a diffraction-limited illumination spot.
  • An Airy is thus an extension of a diffraction disk in an image plane, which is caused by a point in the sample plane.
  • the extent can be defined as a distance between the first zeros of the diffraction disk.
  • a Diffraction-limited light distribution the size of an Airy has a radius of 0.61 ⁇ / ⁇ . Where ⁇ is the wavelength of light and NA is the numerical aperture.
  • the distance between adjacent detector elements may be less than half or one-third Airy disk.
  • One point of the sample plane is thereby always mapped to several neighboring detector elements.
  • FIG. 1 There is shown schematically a sample along the x-axis of a sample plane.
  • the sample comprises a sample point 42 or a fluorescent object 42.
  • a lighting spot 44 is shown. Its intensity I is indicated on the ordinate.
  • the dimensions of the illumination spot 44 can be diffraction-limited and are larger in the x-direction than the object 42. If the illumination spot 44 hits the object 42, it is excited to fluoresce and emits sample light, which can be detected by a detector device.
  • FIG. 1 also shows an illustration of such a detec- tor device 60 in the sample plane, in this case infinitely sharp.
  • the detector device 60 comprises a plurality of detector elements 63, 64. These not only receive sample light emitted by a point in the sample plane. Rather, an extended receive range is imaged onto each detector element, which is determined by the PSF (Point Spread Function) of the image.
  • the PSF for the detector element 64 is shown as a dashed curve 46.
  • the dimensions of the illumination spot 44 can also be determined by a PSF of a point light source.
  • the measured light intensity of a particular detector element 64 is now determined by a total PSF which is the product of the PSF to the illumination spot 44 and the PSF 46 to the detector element 64.
  • the maximum of the total PSF is approximately midway between the illumination spot 44 and the PSF 46 of the respective detector element 64. In the illustrated example, therefore, the detector element 64 receives light primarily from a location 61A centered between the illumination spot 44 and the PSF 46 lies. On the other hand, the detector element 64 hardly measures light from the position 61 D, although at this point the associated PSF 46 has its maximum.
  • the illumination spot is now moved from position 44D to, for example, 44B. This is referred to as illumination scanning. This shifts the total PSF of the detector element 64. This now measures no longer light from mainly the position 61A, but 61 B. This circumstance can be used to increase the resolution.
  • Detector elements to each position of the illumination spot 44 read out.
  • the sample light signals measured thereby are assigned to different sample areas depending on the position of the illumination spot 44. That is, the sample light signals measured by one and the same detector element are resorted depending on the position of the illumination spot 44.
  • the resorting is illustrated by the curved arrows.
  • a signal from detector element 64 is associated with location 61A of object 42 when an illumination spot is at location 44D.
  • a signal of the detector element at the location 61 C is assigned to the location of the object 42 at an illumination spot at the location 44C.
  • a signal of the detector element 61 B is assigned to the location of the object 42 at an illumination spot at the location 44B.
  • the equipment required to achieve this sorting is high.
  • a time required to calculate the resorting is relatively large.
  • the improvement in resolution can also be described as a stronger weighting of the higher spatial frequencies in the optical transfer spectrum of a single-spot system. Because the light distribution within a 1- Airy pinhole diameter is used for imaging, more photons can be detected. The signal-to-noise ratio is thus improved.
  • a laser scanning microscope uses a lighting spot as structured illumination.
  • an increased resolution is achieved by a confocal image, for which a pinhole, ie a pinhole, is positioned in or on an image plane.
  • the signal-to-noise ratio is comparatively low, since only a relatively small amount of light is used.
  • a microscope with a Nipkow disc can be used for simultaneous examination of several sample areas.
  • Such microscopes are described in US 5,428,475 A and US 2008/0218849 A1.
  • By arranging the Nipkow disk in the common illumination and detection spiral path extra-focal light is filtered. Fast image acquisition can be achieved with this relatively simple structure by rotating the Nipkow disk. This is therefore also referred to as a spinning disk.
  • the simultaneous examination of several pinholes of the Nipkow disc a so-called multi-spot examination, can further accelerate the sample examination.
  • a microscope with this structure is described in EP 1 359 452 A1.
  • the object is achieved by a light microscope having the features of claim 1 and a microscopy method having the features of claim 13.
  • the light microscope of the above type is inventively characterized in that a scanning device is provided with at least a first and a second optical arrangement.
  • the optical assemblies of the scanner are simultaneously movable in a common direction to produce an illumination scan and a detection scan that are opposite to each other.
  • the illumination scanning movement is a scanning movement of illumination light over the sample plane, and as detection scanning movement, reception areas of the detector elements can be moved over the sample plane.
  • the first and the second optical arrangement each have a plurality of juxtaposed optical elements with which spaced-apart sample areas can be examined simultaneously.
  • the first and the second optical arrangement are arranged such that both a beam path of the sample light from the sample plane to the detector device and a beam path of the illumination light from the light source to the sample plane extend over the first optical arrangement and only one of these two beam paths via the second optical arrangement runs.
  • sample light is non-inverting and can be imaged with a magnification of less than one with the optics orders of the scanning device.
  • the microscopy method of the aforementioned type is inventively characterized in that optical arrangements of a scanning device are moved simultaneously in a common direction for generating a Beleuchtabtabtastbe- movement and a detection scanning movement, which are opposite to each other.
  • a detection scanning movement reception areas of the detector elements are moved over the sample plane.
  • the first and the second optical arrangement which each have a plurality of juxtaposed optical elements, spaced apart sample areas are examined simultaneously, wherein the first and the second optical arrangement are arranged so that over the first optical arrangement illumination light and sample light are passed and that on the second Optics arrangement only either illumination light to the sample plane or sample light is passed to the detector device out.
  • sample light is imaged in a non-inverting manner and with a magnification of less than one with the optical arrangements of the scanning device.
  • the microscopy method according to the invention is carried out with a microscope according to the invention.
  • the illumination light of optical elements of the first and / or second optical arrangement is split into partial beams, which project onto spaced-apart samples. ben Schemee be routed. Sample light is emitted from the illuminated prism areas. This is forwarded as a partial beam from the optical elements of the first and / or second optical arrangement to the detector unit.
  • the sample area from which a particular detector element receives the largest amount of light depends on the position of the illumination pattern or illumination spot on the sample. This has been explained in more detail with reference to FIG. 1.
  • the illumination scanning movement shifts the illumination pattern on the sample. This also shifts the overall PSF and thus the sample area, from which a particular detector element receives the largest amount of light.
  • it can be achieved by the detection scanning that a particular detector element receives light mainly from the same sample area.
  • the location of the maximum of the total PSF is therefore hardly changed as much as possible by the illumination and detection scanning movement.
  • the detection scanning movement must be opposite to the illumination scanning movement and must take place simultaneously with it.
  • the detector element 64 mainly receives light from the region 61A.
  • the total PSF has its maximum.
  • the illumination spot 44 is then moved in the direction of the arrow 81, for example, until its maximum lies at the position 44C.
  • the detection range of the detection element 64 is moved in the opposite direction as the detection scanning movement, ie in the direction of the arrow 82.
  • the reception range of the detection element 64 can be viewed as the extent of its PSF 46 up to the first minimums of the PSF 46.
  • the sorting described in the prior art can advantageously be avoided in which the received signals of a specific detector element are assigned to different sample positions as a function of the position of the illumination spot.
  • a direction indication of the illumination scanning movement corresponds to the direction in which the partial light bundles of the illumination light move on the sample.
  • the detection scan motion is a movement of the receive area of a particular detector element in the sample plane.
  • the reception area of a detector element is that area in the sample plane from which the detector element receives light.
  • the reception range is determined by the PSF of the image between the sample plane and the image plane and by the dimensions of the associated detector element.
  • a reception area can also be understood as an image of the associated detector element in the sample plane.
  • the illumination scanning movement and the detection scanning movement are opposite when the reception areas of the detection elements in the sample plane are moved opposite to the partial beams of the illumination light.
  • optical arrangements can be considered which, when moved together in a common direction, produce an illumination scanning movement and a detection scanning movement opposite thereto. For this it is first necessary that not all optical arrangements are used both for illuminating the sample and for detecting sample light. Rather, only one of the optical assemblies directs both sample light in the direction of the detector device and illumination light in the direction of the sample plane.
  • This optical arrangement may expediently be the first optical arrangement which is arranged in the beam path closer to the sample plane than the second optical arrangement.
  • the second optical arrangement is used solely for directing illumination light onto the first optical arrangement and on to the sample plane.
  • sample light does not reach the second optical arrangement or, in any case, sample light which is conducted to the detector device.
  • the second optical assembly is used solely for directing sample light toward the detector device, while illuminating light is not directed onto the second optical assembly on the way to the sample plane.
  • sample light is imaged with the optical arrangements not inverting and with a magnification of ⁇ 1.
  • the requirement of a complex coordination between the illumination scanning movement and the detection scanning movement is then eliminated.
  • the optical assemblies each have a plurality of optical elements. Illumination light is then irradiated simultaneously to a plurality of optical elements of the first optical arrangement. Each of the irradiated optical elements then forwards a partial beam. The different partial beams are directed to non-overlapping sample areas. Thus, a multi-spot lighting is provided. By moving the optical assemblies used for the illumination light, the partial beams are shifted, thus producing the illumination scanning movement.
  • a plurality of mutually non-overlapping sample areas are imaged onto different areas of the detector device.
  • the number of simultaneously examined sample areas corresponds exactly to the number of optical elements of the first optical arrangement, which is irradiated simultaneously by the illumination light.
  • the speeds of the detection scanning movement and the illumination scanning movement are equal in magnitude. This is determined by the imaging scale with which the optical assemblies image sample light. For same-rate velocities, the magnification is 1: 2.
  • the optical elements of the first or second optical arrangement can each have a light-collecting effect for non-inverted imaging, and the optimum see elements of the other optical arrangement each have a light-scattering effect.
  • the optical elements of the first optical arrangement can each have a light-collecting effect and the optical elements of the second optical arrangement each have a light-scattering effect.
  • the optical elements of the first optical arrangement with the associated optical elements of the second optical arrangement can each form a Galilean telescope.
  • Associated optical elements of different optical arrangements should in each case be understood as meaning those optical elements which forward the same partial beam of the sample light.
  • the optical elements of the first optical arrangement can have a light-scattering effect.
  • a non-inverted, virtual image of the sample light can be achieved.
  • the illumination light can be passed in this case as a parallel beam on optical elements of the second optical arrangement and on to the first optical arrangement.
  • the optical elements of the second optical arrangement can have a light-collecting effect and a focal length which is shorter than that of the light-diffusing optical elements of the first optical arrangement. As a result, illumination light is focused in an intermediate image plane.
  • the scanning device can also have further optical arrangements, which are arranged in the beam path of the sample light and at the same time are movable in a common direction.
  • one or more image field rotators may be present, for example Abbe-König prisms.
  • the scanning device can have a third and a fourth optical arrangement as image field rotators, which each have an optical element for each of the optical elements of the first optical arrangement.
  • the optical elements of the third optical arrangement together with the respectively associated optical elements of the fourth optical arrangement form Kepler telescopes. These generate an inverted image and thus serve as image field rotators.
  • the optical elements of the first optical arrangement can also be designed as a Kepler telescope together with the associated optical elements of the second optical arrangement.
  • the optical elements of the first and second optical systems each have a light-collecting effect.
  • the pinhole arrangement can be provided.
  • the pinhole diaphragm arrangement preferably has a pinhole diaphragm for each optical element of the first optical assembly.
  • the optical arrangements of the Abtastein direction can have any shape.
  • the optical elements of an optical arrangement can in principle be positioned arbitrarily to one another and the movements of the optical arrangements can take place in any desired common direction.
  • the optical arrangements are each carried out by a rotatable disk.
  • the illumination light is directed to a portion of one of the rotatable discs.
  • the illumination scanning movement thereby takes place along a circle segment and in the direction of rotation.
  • the detection scan moves along a circle segment opposite to the direction of rotation.
  • the rotatable discs are preferably mounted on a common drive shaft. It can also be provided mutually different drive shafts, which are driven by a common motor.
  • the optical elements can be arranged spirally on the rotatable disks, in particular as Archimedean spirals.
  • an adjusting device for linear displacement of the optical assemblies is present.
  • piezoelectric actuators be used.
  • the optical elements of an optical arrangement can also be positioned in a checkerboard pattern.
  • the optical elements of the optical arrangements can in principle be of any type as long as they have a light-collecting or light-scattering effect.
  • the optical elements of the various optical arrangements can, for example, each be formed by at least one lens, a mirror or a light-diffractive element.
  • As light-diffractive elements Fresnel lenses can be used.
  • a relatively simple beam path can be achieved if all the optical elements are formed by lenses.
  • a beam splitter can be arranged between the first and second optical arrangement. This passes illumination light through the first optical assembly, without the illumination light before passing through the second optical arrangement. At the same time, the beam splitter allows sample light coming from the first optical arrangement to be transmitted at least partially to the second optical arrangement.
  • the lenses can be embodied as achromats or ashpears and, in principle, also consist of one or more lens groups.
  • the optical elements of the first optical arrangement can also be lenses and those of the second optical arrangement can be mirrors. Sub-beams of the sample light reflected by the second optical arrangement can then be directed via a further beam splitter in the direction of the detector device.
  • Mirrors can also be used for the optical elements of the first optical arrangement if, for example, a further beam splitter is provided.
  • a preferred value of the above-described magnification of the optical arrangements is 0.5. This value is particularly useful when the PSF, which maps a point light source into the sample plane, and the PSF that images a sample point have the same width. This is the case, for example, in curves 44 and 46 of FIG. As a result, the speeds or increments of the illumination and the detection scanning should be the same amount. This is currently achieved by a magnification of 0.5 optics of the scanner optics. But if the two PSF have different widths or shapes, a different magnification may be preferred be. This may be the case in particular when the wavelengths of the sample light and the illumination light are different from each other, for example in fluorescence measurements.
  • the scanning device may have a zoom optics arrangement. This is movable together with the first optical arrangement and arranged so that it is run through in operation solely by the sample light.
  • the zoom optics assembly may each have one zoom optics per optical element of the first optics assembly.
  • illumination and detection scanning movements are performed during an integration time of the detector elements.
  • the detector elements are therefore not read separately for different positions of the optical arrangements, as is required for sorting according to the prior art. Instead, the detector elements can integrate integrated sample light signals for the acquisition of a sample image while the optical arrangements of the scanning device are being moved.
  • the electronic means are preferably set up for automatic execution of the method variants described above.
  • FIG. 3 shows components of an embodiment of a light microscope according to the invention, wherein the scanning device is in a specific position;
  • FIG. 4 shows the components of FIG. 3 with the scanning device in a different position than in FIG. 3;
  • FIG. 3 shows components of an embodiment of a light microscope according to the invention, wherein the scanning device is in a specific position;
  • FIG. 4 shows the components of FIG. 3 with the scanning device in a different position than in FIG. 3;
  • FIG. 6 shows the components of FIG. 5 with the scanning device in a different position than in FIG. 5;
  • FIG. 7 shows components of yet another embodiment of a light microscope according to the invention, wherein the scanning device is in a specific position
  • Fig. 8 shows the components of Fig. 7, wherein the scanning device is in a different position than in Fig. 7.
  • FIG. 2 schematically shows an exemplary embodiment of a light microscope 100 according to the invention.
  • this comprises a light source 10 for emitting illumination light 15, a sample plane 40 in which a sample 41 to be examined can be positioned, a detector device 60 for detecting sample light 45 and a scanner 50.
  • the scanning device 50 With the scanning device 50, an illumination scanning movement of the illumination light 15 via the sample plane 40 takes place. In addition, the scanning device 50 displaces receiving regions in the sample plane 40, from which certain detector elements 61, 62 of the detector device receive sample light. This movement is called a detection scan. Due to the special design of the scanner 50, the illumination scanning movement and the detection scanning movement are always opposite to each other.
  • the light source 10 may comprise a plurality of laser modules. From this emitted illumination light is guided via optical fibers to a mirror staircase 11. Through this, the beam paths of the laser modules are combined in a common Strahiengang. The illumination light 15 is then directed to the beam splitter 17 via an acousto-optic tunable filter (AOTF) 2, a polarization variator 14 and a telescope for beam expansion 16.
  • AOTF acousto-optic tunable filter
  • the resolution is influenced by the polarization of the illumination light.
  • linear polarization a higher resolution is achieved perpendicular to the polarization direction than parallel to it.
  • circular polarization in the lateral resolution direction independent and medium high.
  • the polarization variator 14 the polarization can be adjusted in the desired manner. For example, several images of the sample can be taken in succession with different polarization directions. These images can then be calculated into a single image with increased resolution in each lateral direction.
  • a common beam axis for illumination and sample light is generated between the sample plane 40 and the beam splitter 1.
  • Illumination light 15 is reflected at the beam splitter 17 at least partially in the direction of the sample plane 40.
  • Sample light 45 is transmitted at the beam splitter 17 at least partially in the direction of the detector device 60.
  • the beam splitter 17 may be designed as a neutral divider. In order not to unnecessarily attenuate the lower-intensity sample light, the beam splitter 17 preferably transmits more than 60% of the incident light.
  • the beam splitter 17 can also be embodied as a color splitter, which transmits or reflects light in a wavelength-dependent manner.
  • the beam splitter 17 can also transmit or reflect polarization-dependent light, which also ensures that sample light 45 is largely transmitted and illumination light 15 is largely reflected.
  • the polarization-dependent beam splitter 17 can also be rotatable.
  • the first optical assembly 51 is here as a rotating disk be executed and includes a plurality of optical elements 71.
  • the optical elements 71 are in the example shown lenses that focus the illumination light 15 in an intermediate image plane 70. As the illumination light 5 strikes several optical elements 71, the illumination light 15 is forwarded in the form of a plurality of mutually spaced-apart partial beams.
  • a pinhole arrangement 55 is present in the intermediate image plane 70.
  • This is likewise designed as a rotatable disc and comprises a plurality of pinhole apertures 75, through which the partial beams of the illumination light 15 are passed.
  • the size of the pinholes is chosen so that the focused partial beams of the illumination light 15 can pass completely or be trimmed at the edge.
  • a pattern of the illumination light 15 in the intermediate image plane 70 is then imaged in the sample plane 40.
  • a tube lens 20, beam deflection means 21 and a lens 30 are provided.
  • the Mafistabsfak- tor which is determined by the focal lengths of the tube lens 20 and the lens 30, can be selected in principle according to the intended investigation in principle.
  • the sample 41 is illuminated by partial beams of the illumination light 15 and emits sample light 45 as a result.
  • This can be scattered illumination light or luminescent light, ie fluorescent or phosphorescent light.
  • the sample light is imaged by the lens 30 and the tube lens 20 in the Eisenbuchebne 70.
  • the pinholes 75 located there leave sample light 45 largely out of the focal plane of the sample 41, while largely filtering out sample light 45 which does not originate from the focal plane. The degree of this suppression depends on the hole diameter.
  • the sample light 45 also includes a plurality of partial beams emanating from the different moistened areas of the sample 41.
  • the portions of the partial beams of the sample light 45 passing through the pinhole apertures 55 are collimated with the lenses 71 of the first optical assembly 51.
  • they can as parallel radiation beams are transmitted through the beam splitter 17 and directed to optical elements 72 of a second optical array 52 of the scanning device 50.
  • the optical elements 72 in the illustrated example are lenses and positioned so that each partial beam of the sample light 45 strikes another optical element 72.
  • optical elements 72 are critical to the opposite directions of illumination and detection scanning motion. This will be described in more detail later.
  • the second optical arrangement 52 is followed by focusing optics 79 with which an image of the sample 41 is generated on the detector device 60.
  • the detector device 60 comprises a plurality of detector elements 61, 62, wherein each partial beam of the sample light 45 impinges on a plurality of detector elements. Some detector devices 60 require darkness during readout of the detector elements. Therefore, the AOTF 12 may be driven to decrease or zero the intensity of the illumination light 15 as the detector elements are read out.
  • the first optical assembly 51 and the pinhole assembly 55 are rotated together.
  • the partial beam originating from an optical element 71 is shifted in this way, which is called an illumination scanning movement.
  • the second optical assembly 52 is rotated in a common direction.
  • these three rotatable discs 51, 52, 55 are mechanically rigidly coupled together on a common drive shaft 65.
  • FIG. 3 schematically shows components of the light microscope from FIG. 2. Shown is a beam path of the illumination light 15 from the beam splitter 17 to the intermediate image plane 70 and a beam path of the sample light 45 from the intermediate image plane 70 to the detector device 60. While the two optical arrangements 51, 52 comprise a multiplicity of optical elements 71 and 72, as shown in FIG. 2, FIG. 3 alone shows the beam path for a partial beam bundle of the illumination light 15 and the sample light 45.
  • the beam path shown applies in the same way to the remaining optical elements 7, 72, as shown in FIG be illuminated. It should be noted that Figure 3 is not to scale.
  • the beam splitter 7 is sufficiently large that reflected illumination light 15 from the beam splitter 17 can reach a plurality of juxtaposed optical elements 71.
  • the focusing optics 79 is also sufficiently large that a plurality of partial beams of the illumination light 45 can be directed onto the detector device 60 with the latter.
  • the optical element 71 comprises a condenser lens and the optical element 72 comprises a diverging lens.
  • the lenses 71, 72 produce a non-inverted virtual image of the intermediate plane 70, from which the focusing optics 79 forms a real image on the detector device 60.
  • the focusing optics 79 can not map either inverted or inverted.
  • the converging lens 71 focuses the illumination light 15, which impinges on the converging lens 71 as a parallel beam bundle, into the intermediate image plane 70.
  • An intensity profile of the illumination light in the intermediate image plane 70 is shown as a curve 44.
  • the image of a sample point 42 in the intermediate image plane 70 is shown.
  • the maximum of the intensity curve 44 of the illumination light 15 is located exactly at the sample point 42.
  • an illustration 160 of the detector device 60 which is infinitely sharp here, is shown.
  • the detector elements 61, 64 are imaged in the intermediate image plane 70 in the positions 161 and 164.
  • FIG. 4 shows the components of FIG. 3 at a different time.
  • the optical elements 71, 72 of the scanning device were moved in a common direction.
  • the other components are stationary.
  • the intensity distribution 44 of the illumination light 15 in the intermediate plane 70 is also shifted upward. Accordingly, the maximum of the intensity distribution 44 no longer lies at the sample point 42, but above it.
  • the path of the displacement of the intensity distribution 44 in the intermediate image plane 70 is equal to the path of movement of the lens 71, because with this the illuminating light 15, which initially extends as a parallel beam, is focused onto an area which is located on a central or optical axis 77 of the lens 71 is located.
  • the stationary detector element 61 in FIG. 4 is located below the central axis or optical axis 77, which runs centrally through the lenses 71, 72. Because the lenses 71, 72 do not image in an inverting manner, an image 161 of the detector element 61 is likewise located below the optical axis 77.
  • the image 161 or the location 161 can also be regarded as the center of the reception area of the detector element 61. Because an image is not generated in an infinitely sharp manner, the detector element 61 also receives light from an extended area around the location 161. This reception area may be defined as an Airy disk on which the detector feature 61 is imaged in the intermediate image plane 70.
  • the detector element 61 would also be imaged on the sample point 42 in the intermediate image plane 70 in FIG. The magnification of the lenses
  • the light-diffusing lens 72 may have a focal length of magnitude half as large as that of light-collecting lens 71 is.
  • the two lenses 71, 72 can also be called Galilei telescopes.
  • a receiving area of a specific detector element in the intermediate image plane 70 thus shifts downward.
  • the displacement of the detector element image 16, which may also be considered as a displacement of the center of the reception area of the detector element 61, may be referred to as a detection sweep.
  • a detection scanning movement downwards is made from FIG. 3 to FIG. 4, compare positions of the images 161.
  • the illumination amplitude movement that is to say the shift of the intensity curve 44, was upward.
  • the light intensity received by the detector element 61 is determined by a total point spread function whose maximum lies between the intensity curve 44 of the illumination light 15 and the location of the image 161.
  • the maximum of the total point spreading function should be as stationary as possible during an illumination scanning movement by the simultaneous detection scanning movement.
  • the lenses 71, 72 preferably produce an image with a scale of 1: 2.
  • the sample image taken by the detector device 60 during the illumination and detection scanning movements is not about half as large as the specimen image in the intermediate plane 70 Rather, it is the same size, as long as a possibly existing focusing optics with a scale of 1: 1 images.
  • the intermediate biaxial plane 70 is not uniformly illuminated with illumination light. Rather, the area of the intermediate biofilm level 70, from which a particular detector element receives the largest amount of light, also depends on the location of the illumination spot 44 in the intermediate biofilm level 70.
  • the illumination and detection scanning movement therefore achieves, at a reproduction scale of 1: 2, that the image of the sample recorded by the detector device 60 has the same size as the image of the sample in the intermediate biilayer 70.
  • the optical elements 71, 72 are moved at the same speed in a common direction. Therefore, high speeds of the scanning movements can be achieved with simple apparatus means.
  • the optical elements 7, 72 are formed with mirrors or diffractive elements instead of lenses.
  • the optical element 71 may be a lens and the optical element 72 may be a mirror. In this case, sample light reflected by the mirror 72 can be conducted in the direction of the detector device 60 with another beam splitter.
  • the optical element 71 may be a mirror.
  • a first beam splitter is used to guide illumination light, which is reflected by the mirror 71, to the sample.
  • Sample light can be conducted via the first beam splitter to the mirror 71 and then with a second beam splitter in the direction of the optical element 72nd
  • a mirror 71 can also be provided with a hole in the middle, whereby the pinhole arrangement can be replaced.
  • FIGS. 3 and 4 can also be used with optical arrangements which are not designed as rotatable disks. Rather, any other one or two-dimensional arrangements of optical elements may be provided. These arrangements need not be rotated, but can be moved together in any manner, for example linear or in a zig-zag form.
  • FIGS. 5 and 6 Another embodiment of a light microscope 100 according to the invention will be described with reference to FIGS. 5 and 6.
  • the light microscope may correspond to that of FIG. 2, wherein between the optical arrangement 52 and the detector device 60 there are two further optical arrangements each having a plurality of optical elements. In this case, the focusing optics 79 can be omitted.
  • the two further optical arrangements like the first and second optical arrangement 51, 52, can be designed as disks which can likewise be rotated by the drive shaft 65.
  • FIG. 5 shows the rays of a partial ray bundle of the illumination light 5 and of a partial ray bundle of the sample light 45. As in FIGS. 3 and 4, the illumination light 5 is focused by a light-collecting lens 71.
  • the sample light 45 is here imaged on the detector device 60 via the lenses 71 to 74.
  • the lenses 71 to 74 each have a light-collecting effect. This allows the lenses 71, 72 to form a first Kepler telescope and the lenses 73, 74 a second Kepler telescope.
  • the first Kepler telescope generates an inverted image, which is again inverted by the second Kepler telescope.
  • the optical elements 71 to 74 of the scanning device generate a non-inverted image.
  • Figure 6 shows a situation in which the lenses 71 to 74 of the scanner have been moved upwards. As a result, an illumination scanning movement and a detection scanning movement are generated in the intermediate image plane 70.
  • the Beschretbonnet apply to the illumination and Detektionsabtastterrorism between Figures 3 and 4 for the embodiment of Figures 5 and 6 accordingly.
  • Figures 7 and 8 show an embodiment in which Beieuchtungslicht 15 is passed via optical elements 72 of the second optical arrangement to optical elements 71 of the first optical arrangement and on to the intermediate image plane 70.
  • the optical element 72 is a condenser lens and the optical element 71 is a diverging lens.
  • the diverging lens 72 generates a non-inverted virtual image with the sample light 45.
  • the sample light 45 is directed to the detector device 60 without going to the converging lens 71.
  • the sample light 45 that is, the partial beams of the sample light 45, which are passed through the different diverging lenses 71, imaged onto the detector device 60.
  • the lenses 71, 72 have been moved downwards in FIG.
  • the illumination scanning movement is thus also carried out in the intermediate biofilm plane 70 downwards. It can be seen, however, that the detection scanning movement has taken place upwards.
  • the specific number of optical elements 71 to 74 and therefore optical assemblies 51, 52 is not critical. Rather, it is decisive that the movable optical elements 71 to 74 of the scanning device 50, which guide the sample light 45 to the detector device 60, generate a non-inverted image.
  • This mapping may be virtual, as in FIGS. 3 and 4, and FIGS. 7 and 8, or real, as in FIGS. 5 and 6. Any subsequent imaging by focusing optics 79 may take place in an inverting or non-inverting manner.
  • the magnification of the entire image generated by the optical elements 71 to 74 of the scanner must be less than 1. At a magnification of greater than 1, the illumination and detection scanning would occur at different speeds in the same direction. Only from a magnification of less than 1, the directions are opposite.
  • the magnification is preferably 0.5, whereby the illumination and the detection scanning movement are opposite in magnitude and equal in their speed.
  • the detector elements can further integrate a received signal while moving the optical elements of the scanner. In contrast to the prior art, it is therefore no longer necessary to read the detector elements separately for each measuring position of the optical elements of the scanning device.
  • the detector elements can be continuously integrated until a scan has been completed with all the optical elements of the optical arrangements.
  • the image or raw image subsequently read has a particularly high resolution without the need for further clearing measures.
  • the re-sorting of the received signals which has been described with reference to the prior art omitted.
  • a high-resolution image of the sample can be recorded in a particularly short time.
  • the illumination light 15 concentrated to a point by a lens (not shown) is coupled into the arrangement by the flattened tip of a retroreflector prism 78.
  • the divergent light emanating from the coupling point is deflected by a beam splitter 17 after Koiiimation by 79.
  • each of the micromirrors On the radial area of the disk, which is swept by the illuminated field during the rotation, there is an arrangement of focusing micromirrors (shown schematically), the center of each of which has an opening 81 which transmits the light.
  • a small portion of the light falling on each of the micromirrors (corresponding to the area of the passage opening in relation to the total area of the respective concave mirror) passes through the mirror through the first impingement and from there passes through the microscope beam path (not shown).
  • the majority of the light is reflected back by the micro-concave mirrors 80, with a focus pattern rotating in the focal plane of the micro-concave mirrors forming a focus pattern.
  • the focus pattern lies in or near the (front) focal plane of the optics 79, it is imaged by the latter in the infinite and reaches the prism 78, where it is reflected again.
  • the nonreflecting, flattened tip of the prism 78 corresponds to the image of the central opening of each of the micro hollow mirror 80.
  • the prism 78 is appropriately advanced a little out of the focal plane of the optic 79, the telecentric foci each fall onto the transparent through holes in the center of the micro-cavities 80, are transmitted therethrough and directed to the sample via the microscope beam path as a spinning dot pattern.
  • the light returned by the sample (by reflection or fluorescence) is filtered through the apertures 81 spatially filtered by the beam splitter 26 and then imaged onto the detector with a further optical arrangement.
  • the task of the prism 78 can also take over a mirror, which then can not be placed in the focal plane of the optics 79 (this would mean that the micromirrors and their image would turn relative to each other in a spotted manner), but only behind another projective in or near the plane of a plane mirror generates an image of the rotating focus pattern of the Mikrospiegei. Again, the mirror must be slightly moved out of the focus plane of the optical arrangement, so that the image of the focus pattern of Mikrospiegei 80 comes to rest in the through holes. Furthermore, a point analogous to the flattened tip of the prism 78 must be created, via which the illumination beam, which is only spot-sized, can be coupled in here. Analogous to the movement of the lenses 71, 72 in FIGS.
  • a shift of the intensity distribution of the illumination light is effected by a common offset of the position of the micromirror 80 and the microdiscuter lens 72 perpendicular to the optical axis to achieve a diametrical displacement between the image of the detector elements and the illumination light.
  • the illumination light 15 concentrated by a lens (not shown) to a point is coupled into the arrangement by the flattened tip of a retroreflector prism 78.
  • the divergent light emanating from the coupling-in point is deflected by a beam splitter 17 after collimation by 79.
  • the prism 78 with its flattened tip were located exactly in the (rear) focal plane of the optics 79, the beam which is sent back to the micromotor 80 in the same way via the beam splitter 17 and optics 79 would be there again. which collimates and reflects back again. If, however, the prism 78 is moved out of the focal plane of the optic 79 in a suitable manner, the telecentric foci each fall onto the transparent through-openings in the center of the microstructure mirrors 80, are transmitted therethrough and are transmitted to the sample via the microscope beam path as a rotating dot pattern directed.
  • the light returned by the sample (by reflection or fluorescence) is filtered through the apertures 81 spatially filtered by the beam splitter 26 and then imaged onto the detector with a further optical arrangement.
  • the task of the prism 78 can also take over a mirror, which then can not be placed in the focal plane of the optics 79 (this would mean that the micromirrors and their image would turn relative to each other in a spotted manner), but only behind another projective In or near the plane of a plane mirror generates an image of the rotating focus pattern of the micromirrors. Again, the mirror must be slightly moved out of the focus plane of the optical arrangement, so that the image of the focus pattern of the micromirror 80 comes to lie in the through holes.
  • a point analogous to the flattened tip of the prism 78 must be provided, via which the illumination spot beam, which is only spot-sized, can be coupled in here.
  • a shift of the intensity distribution of the illumination light is effected by a common offset of the position of the micromirror 80 and the microdiscuter lens 72 perpendicular to the optical axis to achieve a diametrical displacement between the image of the detector elements and the illumination light.
  • AOTF acousto-optic tunable filter
  • 44A-44D show different positions of the intensity distribution 44 of the illumination light

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  • Optics & Photonics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Engineering & Computer Science (AREA)
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Abstract

L'invention concerne un microscope optique comprenant un plan d'échantillon dans lequel un échantillon à examiner peut être positionné, une source lumineuse pour émettre de la lumière d'éclairage, des moyens de reproduction optiques pour guider la lumière d'éclairage dans le plan d'échantillon et un dispositif de détection qui présente plusieurs éléments de détection pour détecter la lumière qui vient de l'échantillon. Des éléments de détection voisins présentent les uns par rapport aux autres une distance qui est plus petite qu'un disque de Ayri et qui génère un point du plan d'échantillon sur le dispositif de détection. Selon l'invention, le microscope optique est caractérisé en ce qu'un dispositif de balayage comprenant au moins une première et une deuxième unité optique est présent, en ce que les unités optiques du dispositif de balayage peuvent être déplacées simultanément dans une direction commune pour produire un mouvement de balayage d'éclairage et un mouvement de balayage de détection qui sont opposés l'un à l'autre, en ce que la première et la deuxième unité optique présentent respectivement plusieurs éléments optiques qui sont disposés les uns à côté des autres et avec lesquels des zones d'échantillon espacées les unes des autres peuvent être examinées simultanément, en ce que la première et la deuxième unité optique sont disposées de manière qu'un trajet de la lumière d'échantillon puisse s'étendre du plan d'échantillon au dispositif de détection et qu'un trajet de la lumière d'éclairage puisse s'étendre de la source lumineuse au plan d'échantillon par l'intermédiaire de la première unité optique et que seul un de ces deux trajets passe par la deuxième unité optique, et en ce que pour atteindre une direction du mouvement de balayage de détection qui est opposée à la direction du mouvement de balayage d'éclairage, la lumière d'échantillon puisse être reproduite non inversée et à une échelle inférieure à un par le dispositif de balayage. L'invention concerne également un procédé de microscopie correspondant.
PCT/EP2014/051301 2013-01-25 2014-01-23 Microscope optique et procédé de microscopie WO2014114702A1 (fr)

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EP14701716.4A EP2948810B1 (fr) 2013-01-25 2014-01-23 Microscope optique et procédé de microscopie
CN201480005722.4A CN104956249B (zh) 2013-01-25 2014-01-23 光学显微镜和显微术方法
US14/762,930 US9671600B2 (en) 2013-01-25 2014-01-23 Light microscope and microscopy method
JP2015554140A JP6286449B2 (ja) 2013-01-25 2014-01-23 光学顕微鏡および顕微鏡観察方法
US15/442,937 US9989746B2 (en) 2013-01-25 2017-02-27 Light microscope and microscopy method

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US9671600B2 (en) 2017-06-06
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US20170192216A1 (en) 2017-07-06
DE102013001238A1 (de) 2014-07-31
US20150378141A1 (en) 2015-12-31

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